Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonizes a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must meet their metabolic demands by coupling to host metabolic processes. While this metabolic dependency is potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co-opted by intracellular T. cruzi amastigotes is lacking.In this broader context, our new discovery that intracellular T. cruzi amastigotes establish close physical contact with host mitochondria via their single flagellum raises the possibility that metabolite sensing and/or acquisition by this intracellular parasite may have a physical basis. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Here, we propose to develop new molecular tools to test the hypothesis that the T. cruzi amastigote flagellum-host mitochondria contacts are mediated by protein- protein interactions and serve an important function in the parasite's intracellular life cycle. To this end, two complementary and innovative strategies are proposed: (1) To identify T. cruzi amastigote flagellar surface proteins as candidate mediators of the host mitochondria association using a targeted proximity-labeling approach for in situ biotinylation followed by mass spectrometry, and (2) To develop an image-based screening platform to identify small molecule inhibitors that disrupt the flagellum-mitochondria interaction to enable functional studies. Results will significantly advance our goal of determining the mechanistic basis for metabolic coupling between intracellular T. cruzi and its mammalian host cell with a long-term view of exploring the translational potential of these novel findings.